Short Communication IXth International Conference on Boar Semen Preservation 2019

The effect of pre-freeze cooling and melatonin on pig sperm plasma membrane fluidity and cryosurvival (#3.1)

Alfredo Medrano 1 , Jessica K Orozco 1 , Daysi A Martinez-Hernandez 1 , Maricela Rafael-Duran 1 , Oscar Gutierrez-Perez 2 , Alicia Alcantar-Rodriguez 1
  1. National Autonomous University of Mexico (FES Cuautitlan), Cuautitlan Izcalli, ESTADO DE MEXICO, Mexico
  2. CEIEPP, National Autonomous University of Mexico (FMVZ), Jilotepec, Estado de Mexico, Mexico

Pig sperm are highly susceptible to cryopreservation protocols; oxidative stress as well as drastic changes in plasma membrane fluidity alters sperm physiology. Use of pre-freeze cooling to sub-zero temperatures and antioxidants are experimental approaches directed to reduce the occurrence of sperm plasma membrane hyper-fluidity, and oxidative stress during cryopreservation. The objective of this work was to assess the effect of pre-freeze cooling to -5°C, and melatonin (MLT) as an antioxidant, on boar sperm plasma membrane fluidity and cryosurvival. After collection, ejaculates were diluted, transported (34 °C, 90 min), centrifuged and the supernatant removed. Only ejaculates showing at least 80% progressive motility and 85% viability of sperm were included in the experiments. In the first, sperm (3 boars, 9 ejaculates) were resuspended in lactose-egg yolk freezing medium (3% glycerol), and packaged in 0.5 mL plastic straws; in the second, sperm (4 boars, 18 ejaculates) were resuspended in BF5 freezing medium (1% glycerol) containing 0, 1 or 2 mM melatonin (MLT), and packaged in 0.25 mL plastic straws. Each ejaculate was split and all treatments were applied to aliquots of one ejaculate. Diluted sperm were cooled from 23 to 5 °C into a common refrigerator over 2 h. One half of the straws at 5 °C was frozen over nitrogen vapours and immersed in liquid nitrogen; the other half was further cooled to -5°C, and then frozen. An insulated box containing crushed saline ice (10% w/v) at −12°C was used to cool the straws to -5 °C [1]. Straws were thawed in a water bath at 39 °C for 30 sec. Sperm were assessed for progressive motility (visual), viability (eosin-nigrosine), plasma membrane integrity (SYBR14/PI) and fluidity (MC540), acrosome integrity (PSA-FITC), and capacitation status (CTC assay). Data was analysed by “t” test and ANOVA. In the first experiment, there were no differences between cooling treatments for sperm quality; MC540 high-binding cells (hyper-fluid membranes) were 52, 57, and 11% for cooling to +5°C, -5°C, and before cooling respectively. In the second experiment, there were differences (P<0.05) between cooling treatments in plasma membrane integrity, non-capacitated acrosome-intact, and capacitated acrosome-intact spermatozoa (+5°C better than -5°C). There were no differences in sperm quality between MLT treatments. MC540 high-binding cells were 34, 32, and 32% for 0, 1 and 2 mM MLT, respectively. In conclusion, percentage of hyper-fluid membranes increased after freeze-thawing regardless of MLT and pre-freeze cooling temperature. Pre-freeze cooling was more important than MLT in boar sperm cryosurvival.

Supported by UNAM (PAPIIT IA204917, IA220419, PIAPI 1615/1649/1810).